Tumor chemoresistance and metastasis are some of the most important problems in colon cancer therapy. In the present study, co-cultures of human colon carcinoma cell spheroids, obtained from different grades of tumor, with human colon epithelium, myofibroblast and endothelial cell monolayers were performed. The purpose of these co-cultures was to reflect, in in vitro conditions, different stages of colon tumor development. In order to investigate the invasive capacities of the tumor cells and their resistance to chemotherapy, HGF, HSP27, HSP72 and MRP levels were analyzed after incubation of the co-cultures with IL-1beta and irinotecan (CPT-11) added as single agents or in combination. Myofibroblasts produced significantly higher amounts of HGF than epithelial cells. Tumor cells released trace amounts of this molecule. In cocultures, IL-1beta induced HGF release, while CPT-11 alone or combined with IL-1beta decreased HGF secretion. An immunoblotting analysis followed by densitometry revealed that the combination of IL-1beta plus CPT-11 added to the cocultures led to a decrease in HSPs and MRP levels. In conclusion, direct and paracrine interactions of colon tumor cell spheroids with normal cells and exogenously added CPT-11 change HSP27, HSP72 and MRP expression in comparison to monocultures. IL-1beta and CPT-11, dependent on whether they are added separately or jointly, differentially modulate HGF expression in monocultures of colon tumor spheroids or normal cells and their co-cultures.